This invention relates to peptide conjugates which can be used to form an immunogenic composition useful to activate the immune system of a patient exposed to or at risk of infection by Herpes Simplex Virus (HSV). More particularly, this invention relates to immunogenic conjugated peptides which contain both a herpes simplex virus specific peptide and an immunomodulatory peptide covalently linked directly or via a linking group and to compositions and diagnostic products containing and methods using such peptide conjugates in the treatment, prevention, or diagnosis of herpes simplex virus.
Herpes simplex virus type 1 (HSV-1) and its close cousin, herpes simplex virus type 2 (HSV-2), cause various benign diseases, such as the common cold sore found near the lips and also genital herpes. Herpes simplex virus can also cause serious disease upon infection of the eye (e.g., keratoconjunctivitis, with the potential to lead to blindness), the brain (e.g., encephalitis). Individuals who are immunosuppressed, such as a newborn baby, AIDS patient or transplant patient, are especially vulnerable. HSV infections of immunocompromised individuals and neonates can lead to disseminated and life-threatening disease. Unlike many viruses, once an individual is infected with HSV, the virus remains latent in neurons and can be reactivated by stress or immunosuppression and cause recurrent disease.
A herpes simplex virus vaccine has the potential for use as both a prophylactic to prevent initial infection as well as a treatment to prevent or ameliorate recurrent disease. No vaccine is currently available for prevention or treatment of HSV. Live vaccine development has been hampered because HSV has the capacity to establish latent infection and potentially, to promote neoplastic transformation of cells. The efficacy of live attenuated killed and subunit vaccines has been questioned due to difficulties in eliciting protective cell mediated immune responses.
Cellular immunity and specifically, cytotoxic T lymphocyte cell (CTL) and delayed type hypersensitivity (DTH) responses, are important for the control of HSV. These responses are included in the TH1-type of immune response initiated by CD4 and CD8 T cells following appropriate activation. CD 8 T cells recognize 8-9 amino acid peptides presented by an MHC I molecule and become the target for CTL killing of infected cells. However, immunization with these and other CTL epitope-containing peptides rarely elicits protective immunity because the peptides are too small to be recognized and presented to the immune system. In order to use such a peptide as a vaccine, the peptide must be modified or presented in a manner which will make it more visible to the immune system. Ideally, the modification or manner of presentation should itself be invisible to the immune response and should facilitate the development of the appropriate T cell and overall immune response to the peptide.
T cell responses can be classified by the cytokine profile produced in response to activation. T1 (TH1:CD4 T cells and TC1:CD8 T cells) responses are associated with production of interferon gamma, interleukin-2, lymphotoxin alpha, preferential stimulation of IgG2a production in the mouse, and induction of DTH reactions. T2 (TH2 and TC2) responses are associated with production of interleukin-4, interleukin-10, preferential stimulation of humoral responses, IgG1 production and inhibition of T1 responses. Stimulation of a T2 response may exacerbate infectious diseases which are controlled by protective T1 responses.
Traditionally, small peptides must be attached to carrier proteins in order to elicit immune responses. Often a large protein such as KLH is used. However, it has been observed that heterogenous (impure) KLH yields a better immune response than a more homogenous preparation. Nevertheless, these conjugates tend to promote the production of T2 associated responses and antibody is developed to both the peptide and the carrier.
It would be desirable to find other methods to direct the response primarily or substantially to murine IgG2a or human IgG3, a TH1 associated pathway. It has previously been proposed by one of the present inventors that the carrier should not be directing the response in an undesired direction and since the KLH molecule seems to be predominantly directing the response in the TH2 direction it was concluded that another carrier should be considered. Likewise, other factors such as costs, ease of manufacture, and stability need to be taken into account.
Previous serological studies with M. tuberculosis peptides conjugated to a T cell binding ligand (TCBL) derived from the xcex2-2-microglobulin protein of the Major Histocompatibility Complex (MHC) Class I (specifically, at amino acid positions aa 38-50, hereinafter referred to as xe2x80x9cxcex2-2M 38-50xe2x80x9d or, alternatively, as xe2x80x9cPeptide Jxe2x80x9d) induced a TH1-like response whereas similar peptide constructs but using a TCBL, such as MHCIIxcex22 135-149 (Peptide G) induced a TH2-like response (Zimmerman, et al., Vacc. Res. 5:103-118, 1995).
Generally, incorporation of an antigenic peptide into a conjugated polypeptide with the appropriate T cell ligand will elicit a defined T cell response. The T cell binding ligand incorporated into the construct will determine whether the response is a TH1 or a TH2 response or mixed or predominantly one or the other of these responses. For any particular disease causing microorganism either or both types of responses may be desirable or preferred. However, elicitation of a TH1 (DTH) type response would be expected to be especially useful against infectious agents which are normally successfully resolved by TH1 (e.g., DTH) responses rather than TH2 responses, associated with mature antibody responses. The conjugated polypeptide technology is effective in allowing small peptides to elicit appropriate TH1 responses (or/and TH2 responses, in appropriate cases) without large protein carriers.
One of the present inventors previously reported that addition of a T cell binding ligand to a peptide epitope could alter the nature of the immune response (i.e., TH1 or TH2). Antibodies derived from certain conjugated polypeptides were better able to recognize the native molecule than were the antibodies prepared by using a conventional peptide-KLH conjugate.
It was shown that the antibodies induced by the heteroconjugate had a broader specificity, so that they recognized the peptide epitope not only in the linear form, but also in the native molecule. In some cases the use of the peptide conjugated to KLH was not able to recognize the epitope in the native molecule. Accordingly, it would be highly desirable to provide a vaccine for HSV which would be effective to prevent initial infection as well as a treatment for individuals who suffer from frequent recurrent disease associated with HSV.
The present invention relates to certain conjugated polypeptides comprising at least two T cell specific binding peptides covalently linked together, wherein the first peptide binds to a specific class or subclass of T cells and elicits a predominantly T1 or T2 type response and a second peptide is an herpes simplex virus specific antigenic peptide, and wherein the polypeptide is capable of eliciting TH1 associated antibodies when administered to a human in need thereof.
The peptide used as T cell specific binding molecule in the conjugated peptides of this invention are peptides which are portions of molecules or analogues of such portions which bind specifically or at least preferentially to specific class or subclass of T cells, such as helper T cells (TH), suppressor T cells (TS), cytotoxic T lymphocytes (CTL), and the like, and which directs the immune system to a predominantly T1 type immune response against the HSV specific antigenic peptide. These conjugated polypeptides offer the advantages previously seen with other conjugated peptides, such as those more generally disclosed by one of the present inventors in U.S. Pat. No. 5,652,342 (referred to therein as xe2x80x9cheterofunctional cellular immunological reagent), of inducing broad spectrum antibodies but, additionally providing a desired T1 specificity.
In particular, the invention provides an immunogenic conjugated polypeptide effective as immunogen in a vaccine for treatment or prevention of infection by herpes simplex virus, said polypeptide represented by the formula P1xe2x80x94xxe2x80x94P2 or P2xe2x80x94xxe2x80x94P1 where P1 represents a herpes simplex virus specific antigenic peptide from a herpes simplex virus type 1 or type 2, selected from the group consisting of the ICP27 protein, glycoprotein B, ribonucleotide reductase, ICP4, ICP34.5, glycoprotein E and glycoprotein F; P2 represents an immunodulatory peptide which is a portion of an immunoprotein which promotes binding to a class or subclass of T cells and which direct a predominantly TH1 type immune response to the peptide P1; and x represents a covalent bond or a divalent peptide linking group, which may be cleavable or non-cleavable.
The present invention also relates to pharmaceutically effective compositions containing such conjugated polypeptide for eliciting immunization to infection against Herpes Simplex Virus, HSV, type 1 or type 2, in a human subject. Such compositions, in addition to the conjugated polypeptides of this invention may also, and preferably will, include suitable immunological adjuvant.
Similarly, the invention relates to the use of such conjugated polypeptide and the pharmaceutically effective composition containing same for treating or preventing HSV infection by administering to a human patient in need thereof, a therapeutically or prophylactively effective amount of the conjugated polypeptide as defined above.
The invention also relates to a diagnostic assay for diagnosing the presence of infection (active or latent) in an individual by HSV wherein T cells from the individual to be diagnosed is mixed with the conjugated polypeptide to the above formula P1xe2x80x94xxe2x80x94P2 or P2xe2x80x94xxe2x80x94P1, and thereafter detecting a reaction between previously HSV primmed T cells and conjugated polypeptide. The conjugated polypeptide may be labelled to facilitate detection of the reaction.
The invention will now be described in further detail by way of the following explanations and preferred embodiments and with the aid of the accompanying drawings.
It is expected that HSV immune T cells would behave in differently or otherwise be different in the presence of the conjugated polypeptide compared to niave or non-immune T cells.